Epithelial Cdc42 Deletion Induced Enamel Organ Defects and Cystogenesis.

Cdc42, a Rho family small GTPase, regulates cytoskeleton organization, vesicle trafficking, and other cellular processes in development and homeostasis. However, Cdc42's roles in prenatal tooth development remain elusive. Here, we investigated Cdc42 functions in mouse enamel organ. Cdc42 showed highly dynamic temporospatial patterns in the developing enamel organ, with robust expression in the outer enamel epithelium, stellate reticulum (SR), and stratum intermedium layers. Strikingly, epithelium-specific Cdc42 deletion resulted in cystic lesions in the enamel organ. Cystic lesions were first noted at embryonic day 15.5 and progressively enlarged during gestation. At birth, cystic lesions occupied the bulk of the entire enamel organ, with intracystic erythrocyte accumulation. Ameloblast differentiation was retarded upon epithelial Cdc42 deletion. Apoptosis occurred in the Cdc42 mutant enamel organ prior to and synchronously with cystogenesis. Transmission electron microscopy examination showed disrupted actin assemblies, aberrant desmosomes, and significantly fewer cell junctions in the SR cells of Cdc42 mutants than littermate controls. Autophagosomes were present in the SR cells of Cdc42 mutants relative to the virtual absence of autophagosome in the SR cells of littermate controls. Epithelium-specific Cdc42 deletion attenuated Wnt/ß-catenin and Shh signaling in dental epithelium and induced aberrant Sox2 expression in the secondary enamel knot. These findings suggest that excessive cell death and disrupted cell-cell connections may be among multiple factors responsible for the observed cystic lesions in Cdc42 mutant enamel organs. Taken together, Cdc42 exerts multidimensional and pivotal roles in enamel organ development and is particularly required for cell survival and tooth morphogenesis.

Insulin-like peptides regulate vitellogenesis and oviposition in the green lacewing, Chrysopa septempunctata.

Insulin-like peptides (ILPs) act through a conserved insulin signaling pathway and play crucial roles in insect metabolism, growth, reproduction, and aging. Application of bovine insulin is able to increase vitellogenin (Vg) mRNA and protein levels in female insects. Here, we first show that injection of bovine insulin into previtellogenic Chrysopa septempunctata female adults promoted ovarian growth, increased Vg protein abundance, elevated reproductive performance, and enhanced protease activity. These data suggested that ILPs play crucial roles in reproductive regulation of the green lacewing, C. septempunctata.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

Soft tissue ossification and condylar cartilage degeneration following TMJ disc perforation in a rabbit pilot study.

OBJECTIVE: There are limited clinical treatments for temporomandibular joint (TMJ) pathologies, including degenerative disease, disc perforation and heterotopic ossification (HO). One barrier hindering the development of new therapies is that animal models recapitulating TMJ diseases are poorly established. The objective of this study was to develop an animal model for TMJ cartilage degeneration and disc pathology, including disc perforation and soft tissue HO. METHODS: New Zealand white rabbits (n = 9 rabbits) underwent unilateral TMJ disc perforation surgery and sham surgery on the contralateral side. A 2.5 mm defect was created using a punch biopsy in rabbit TMJ disc. The TMJ condyles and discs were evaluated macroscopically and histologically after 4, 8 and 12 weeks. Condyles were blindly scored by four independent observers using OARSI recommendations for macroscopic and histopathological scoring of osteoarthritis (OA) in rabbit tissues. RESULTS: Histological evidence of TMJ condylar cartilage degeneration was apparent in experimental condyles following disc perforation relative to sham controls after 4 and 8 weeks, including surface fissures and loss of Safranin O staining. At 12 weeks, OARSI scores indicated experimental condylar cartilage erosion into the subchondral bone. Most strikingly, HO occurred within the TMJ disc upon perforation injury in six rabbits after 8 and 12 weeks. CONCLUSION: We report for the first time a rabbit TMJ injury model that demonstrates condylar cartilage degeneration and disc ossification, which is indispensible for testing the efficacy of potential TMJ therapies.

Both genes and lncRNAs can be used as biomarkers of prostate cancer by using high throughput sequencing data.

OBJECTIVE: To investigate prostate cancer-related genes and lncRNAs by using a high throughput sequencing dataset. MATERIALS AND METHODS: RNA-seq data were obtained from the sequencing read archive database, including both benign and malignant tumor samples. After aligning the RNA-seq reads to human genome reference, gene expression profile as well as lncRNA expression profile was obtained. Next, student's t-test was used to screen both the differentially expressed genes (DEGs) and lncRNAs (DELs) between benign and malignant samples. Finally, goseq was used to conduct the functional annotation of DEGs. RESULTS: A total of 7112 DEGs were screened, such as ZNF512B, UCKL1, STMN3, GMEB2, and PTK6. The top 10 enriched functions of DEGs were mainly related to organism development, including multi-cellular development, system development and anatomical structure development. Also, we discovered 26 differentially expressed lncRNAs. CONCLUSIONS: The analysis used in this study is reliable in screening prostate cancer markers including both genes and lncRNAs by using RNA-seq data, which provides new insight into the understanding of molecular mechanism of prostate cancer.

Physiologic load-bearing characteristics of autografts, allografts, and polymer-based scaffolds in a critical sized segmental defect of long bone: an experimental study.

BACKGROUND: To address the challenge of treating critical sized intercalary defects, we hypothesized that under physiologic cyclic loading, autografts, allografts, and scaffolds loaded with and without human mesenchymal stem cells (hMSCs) would have different biomechanical characteristics. METHODS: Using a rat femoral defect model, 46 rats were assigned to four groups, ie, autograft (n = 12), allograft (n = 10), scaffold (n = 13), and scaffold with hMSCs (n = 11). The scaffold groups used a 5 mm segment of scaffold composed of 80% poly-ε-caprolactone and 20% hydroxyapatite. Rats were sacrificed 4 months postoperatively, and the repairs were assessed radiographically and biomechanically. RESULTS: Autograft and allograft groups exhibited the most bridging callus, while the scaffold/hMSCs group had more callus than the scaffold repairs. Although signs of radiographic healing did not accurately reflect restoration of mechanical properties, addition of hMSCs on the scaffold enhanced bone formation. The scaffold alone group had significantly lower elastic and viscous stiffness and higher phase angles than other repairs and the contralateral controls. Addition of hMSCs increased the elastic and viscous stiffness of the repair, while decreasing the phase angle. CONCLUSION: Further comparative analysis is needed to optimize clinical use of scaffolds and hMSCs for critical sized defect repairs. However, our results suggest that addition of hMSCs to scaffolds enhances mechanical simulation of native host bone.

Induced migration of dental pulp stem cells for in vivo pulp regeneration.

Dental pulp has intrinsic capacity for self-repair. However, it is not clear whether dental pulp cells can be recruited endogenously for regenerating pulp tissues, including mineralizing into dentin. This work is based on a hypothesis that dental pulp stem/progenitor cells can be induced to migrate by chemotactic cytokines and act as endogenous cell sources for regeneration and mineralization. Dental stem cells (DSCs) were isolated from adult human tooth pulp and seeded on the surfaces of 3D collagen gel cylinders that were incubated in chemically defined media with stromal-derived factor-1α (SDF1), basic fibroblast growth factor (bFGF), or bone morphogenetic protein-7 (BMP7). Significantly more cells were recruited into collagen gel by SDF1 or bFGF than without cytokines in 7 days, whereas BMP7 had little effect on cell recruitment. BMP7, however, was highly effective, equally to dexamethasone, in orchestrating mineralization of cultured DSCs. Cell membrane receptors for SDF1, bFGF, and BMP7 were up-regulated in treated DSCs. Upon in vivo delivery, bFGF induced re-cellularization and re-vascularization in endodontically treated human teeth implanted into the dorsum of rats. Thus, endogenous dental pulp cells, including stem/progenitor cells, may be recruited and subsequently differentiated by chemotaxis of selective cytokines in the regeneration of dental pulp.

Stroke after treatment with bortezomib and dexamethasone in a Chinese patient with extramedullary relapse of multiple myeloma.

Thromboembolic complications commonly occur in patients with multiple myeloma (MM). The risk of such complications may be elevated by the use of immunomodulatory agents such as thalidomide and lenalidomide as initial therapy for MM. However, arterial thrombosis after treatment with bortezomib is rare. Herein we report a case of a 70-year-old Chinese male patient with extramedullary relapse of MM. After treatment with bortezomib and dexamethasone he developed a nonfatal thrombotic stroke. Administration of bortezomib and dexamethasone was then discontinued and he obtained partial remission.

Anatomically shaped tooth and periodontal regeneration by cell homing.

Tooth regeneration by cell delivery encounters translational hurdles. We hypothesized that anatomically correct teeth can regenerate in scaffolds without cell transplantation. Novel, anatomically shaped human molar scaffolds and rat incisor scaffolds were fabricated by 3D bioprinting from a hybrid of poly-epsilon-caprolactone and hydroxyapatite with 200-microm-diameter interconnecting microchannels. In each of 22 rats, an incisor scaffold was implanted orthotopically following mandibular incisor extraction, whereas a human molar scaffold was implanted ectopically into the dorsum. Stromal-derived factor-1 (SDF1) and bone morphogenetic protein-7 (BMP7) were delivered in scaffold microchannels. After 9 weeks, a putative periodontal ligament and new bone regenerated at the interface of rat incisor scaffold with native alveolar bone. SDF1 and BMP7 delivery not only recruited significantly more endogenous cells, but also elaborated greater angiogenesis than growth-factor-free control scaffolds. Regeneration of tooth-like structures and periodontal integration by cell homing provide an alternative to cell delivery, and may accelerate clinical applications.

Craniofacial tissue engineering by stem cells.

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

Tissue-engineered neogenesis of human-shaped mandibular condyle from rat mesenchymal stem cells.

The temporomandibular joint is susceptible to diseases and trauma that may ultimately lead to structural degeneration. Current approaches for replacing degenerated mandibular condyles suffer from deficiencies such as donor site morbidity, immunorejection, implant wear and tear, and pathogen transmission. The hypothesis of this study was that a human-shaped mandibular condyle can be tissue-engineered from rat mesenchymal stem cells (MSCs) encapsulated in a biocompatible polymer. Rat bone marrow MSCs were isolated and induced to differentiate into chondrogenic and osteogenic cells in vitro, and encapsulated in poly(ethylene glycol)-based hydrogel in two stratified layers molded into the shape of a cadaver human mandibular condyle. Eight weeks following in vivo implantation of the bilayered osteochondral constructs in the dorsum of immunodeficient mice, mandibular condyles formed de novo. Microscopic evaluation of the tissue-engineered mandibular condyle demonstrated two stratified layers of histogenesis of cartilaginous and osseous phenotypes. The current approach is being refined for ultimate therapeutic applications.